FIG. 5.

TcpR is sufficient for IM localization of TcpT, but stable IM localization of TcpT and the stability of TcpR itself require additional V. cholerae-specific factors. (A) Whole-cell extracts of V. cholerae ΔtcpR, E. coli BL21, or V. cholerae ΔtcpP carrying a TcpR-6xHis construct, grown in the presence of 0.02% arabinose at both 30°C (tcp expression ON in V. cholerae) and 37°C and separated by SDS-PAGE, followed by anti-His immunoblotting. (B) Cytoplasmic (C) and IM fractions from E. coli BL21 expressing the indicated combination of TcpR-PhoA and/or TcpT constructs were separated by SDS-PAGE, followed by anti-PhoA immunoblotting. (C) WT V. cholerae IM fraction (first lane only) and E. coli BL21 cytoplasmic (C) and IM fractions were separated by SDS-PAGE, followed by immunoblotting using anti-TcpT antibody. The black arrows indicate bands corresponding to either TcpT or a putative degradation product of TcpT (indicated as TcpT*), and the white arrows indicate nonspecific bands. (D) SDS-PAGE followed by immunoblotting of the cytoplasmic (C) and IM fractions of a tcpP deletion mutant and a tcpR deletion mutant expressing TcpT and/or TcpR-PhoA grown under TCP-inducing conditions and probed with anti-TcpT (top) and anti-PhoA antibody (bottom).